Diabetes is an epidemic in USA and Veterans are nearly three times as likely as the general population to have diabetes. Patients with diabetes have an increased risk of developing cardiovascular disease and more than 75 percent of them die of cardiovascular complications. Therefore, it is crucial to develop new therapeutic strategies for cardiovascular disease to reduce mortality in diabetic patients. In recent years, the innate immunity (immunity a person is born with) has been shown to play an important role in atherosclerosis, a major cause of cardiovascular disease. Studies have shown that deficiency of toll-like receptor (TLR)4 that triggers cellular innate immune responses is associated with a significant reduction of atherosclerotic lesions in nondiabetic mice, suggesting that TLR4 is involved in atherosclerosis. However, these studies only demonstrated the effect of lack of TLR4 on development of atherosclerosis. From clinical point of view, since it is known that stabilization of atherosclerotic plaques is critical to prevent cardiovascular events, and that all patients have TLR4 expression and many of them had developed atherosclerosis when they were diagnosed with type 2 diabetes, it is critical to determine if TLR4 inhibition with TLR4 antagonist in diabetic animal models that already have atherosclerosis changes plaque composition and gene expression that favor plaque stability. Nevertheless, the information is lacking. SPECIFIC OBJECTIVES AND HYPOTHESES: We found that elevated glucose concentrations (high glucose), a hallmark of diabetes, enhanced lipopolysaccharide (LPS)-stimulated matrix metalloproteinase (MMP) and proinflammatory cytokine expression by mononuclear cells. We also found that high glucose enhanced interferon gamma (IFN3)-stimulated MMP expression by upregulating MD-2, a TLR4-associated protein that confers TLR4 signaling. Since TLR4 is the receptor for LPS, and MMPs and proinflammatory cytokines play a crucial role in atherosclerotic plaque instability, the above findings suggest that TLR4 may be involved in diabetes-promoted plaque destabilization. Furthermore, we found that TLR4 antagonist Rs-LPS (isolated from nontoxic photosynthetic bacterium R. sphaeroides) blocked LPS-stimulated MMP-1 expression by mononuclear cells. Recent studies also showed the potency of Rs-LPS as a TLR4 antagonist. Based on these findings, we hypothesized that TLR4 inhibition with Rs-LPS in mice with atherosclerosis and diabetes or metabolic syndrome changes the lesion composition and gene expression that favor plaque stability. In this project, we will conduct animal studies to test our hypothesis. In addition, we will also conduct in vitro studies to further investigate the mechanisms involved in the stimulation of MD-2 expression by high glucose and IFN3. Two specific objectives were proposed: 1. To test our hypothesis that TLR4 inhibition in mice with established atherosclerosis and diabetes changes the lesion composition and gene expression that favor plaque stability. 2. To determine the signaling mechanisms involved in the stimulation of MD-2 expression by high glucose and IFN3, and the role of MD-2 in the IFN3 signaling. METHODS: We will first induce atherosclerotic lesions in mice with diabetes or metabolic syndrome and then treat mice with TLR4 antagonist Rs-LPS. After treatment, the plaque composition and gene expression will be analyzed. In the in vitro study, we will use macrophages and macrophage cell line and perform molecular biology studies such as electrophoretic mobility shift assay and promoter activity analysis. SIGNIFICANCE: This study will establish whether there is a rationale for targeting TLR4 in prevention of cardiovascular event in patients with diabetes or metabolic syndrome. POTENTIAL IMPACT ON VETERANS HEALTH CARE: The incidence of diabetes in Veterans is much higher than that in general population. This research project will provide important information for developing a new therapeutic strategy to prevent fatal cardiovascular events in our Veterans with diabetes.